The proposed studies will address fundamental questions regarding the mechanisms underlying inflammation-induced enhancement of both hyperactive (contractile) and proliferative (synthetic) states of human airway smooth muscle (hASM), which are hallmarks of asthma. Identifying these mechanisms is the key to developing novel therapeutic targets for asthma. Our central hypothesis is that in hASM, inflammatory cytokines induce sarco-endoplasmic reticulum (SR/ER) stress leading to reduced expression of the mitochondrial fusion protein mitofusin 2 (Mfn2), and that this pathway plays a central role in asthma by triggering both hyper reactive (contractile) and proliferative (synthetic) states. Four Specific Aims are proposed: Specific Aim 1: To determine the impact of inflammatory cytokines on SR/ER stress, Mfn2 expression and mitochondrial fragmentation. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that inflammatory cytokines trigger SR/ER stress due at least in part to an increase in ROS generation, and that SR/ER stress leads to reduced Mfn2 expression and increased mitochondrial fragmentation. Specific Aim 2: To determine the functional impact of SR/ER stress and reduced Mfn2 expression. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that in hASM, inflammatory cytokine-induced SR/ER stress and reduced Mfn2 expression uncouples mitochondria and the SR/ER, thereby reducing mitochondrial Ca2+ buffering leading to elevated [Ca2+]cyt and force responses to agonist stimulation. Specific Aim 3: To determine the impact of SR/ER stress and reduced Mfn2 on hASM cell proliferation. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that inflammatory cytokines increase hASM cell proliferation as a result of decreased Mfn2 expression. Specific Aim 4: To determine the efficacy of therapeutic approaches targeting SR/ER stress in alleviating airway hyper reactivity and remodeling in a mouse model of asthma. In this aim, we will use a mixed allergen mouse model to test the hypothesis that targeting SR/ER stress using chemical chaperones (e.g., 4-PBA, TUDCA) will provide an effective therapeutic strategy to reverse airway hyperactivity and blunt remodeling associated with asthma.